Project Summary This work aims to pioneer the study of early events in neural crest (NC) formation in the rabbit embryo. NC is a vertebrate-specific multipotent cell population which forms during early development and migrates extensively throughout the body, contributing to a variety of derivatives including melanocytes, peripheral nervous system neurons and glia, and craniofacial bone and cartilage. Aberrant NC development results in a host of human health conditions, including frequent malformations such as cleft lip and cleft palate, rare syndromes, and aggressive cancers. Studying NC biology is of utmost relevance if we are to ameliorate the effects of such conditions through diagnostic and therapeutic efforts. Considerable progress has been gained towards our understanding of NC development though studies in animal models including Xenopus, zebrafish, chick, and mouse embryos. Recently, studies in Xenopus and chick suggest that early events in NC development initiate around or prior to gastrulation; however, specific knowledge of their formation and early events in mouse and mammalian embryos are lacking. Furthermore, molecular models of NC development cemented on non- mammalian studies remain disputed because equivalent functions/requirements in mammals have not been found. Despite the important role and benefits of the mouse as a model for mammalian development, it has become apparent that the requirement for some key NC genes (e.g. Snai1/2) in non-mammalian model systems are not recapitulated in mouse studies. Our laboratory and others are using human embryonic stem cell-based models of human NC to both interrogate their formation and differentiation potential, empowering hopes for translational efforts. However, it remains difficult to ascertain the degree to which these models recreate normal embryonic development. This leaves open major unanswered questions surrounding mammalian NC formation, and underscores a critical need for the use of additional mammalian model systems. Here, we propose to characterize and interrogate early rabbit NC development for the first time, providing simultaneously, a first account of these events in a mammalian embryo. The early rabbit embryo develops as a large, flat blastodisc similar to human, and displays kinetics of embryonic gene activation and placental development that closely resemble human processes. The developmental and physiological characteristics of the rabbit make it an excellent candidate for translational biomedical research, and a sequenced genome and successful transgenic and knockout approaches place the rabbit in a prime position to be more broadly used for early mammalian developmental studies. Our aims are: (1) to characterize the expression of genes related to NC during early rabbit development (including novel candidates identified from our stem cell-based human NC model); (2) to interrogate the specification and induction of rabbit NC; and (3) to assess the requirement of Snai1 and Snai2 genes for rabbit NC development. This innovative work will unveil novel information about the specification and formation of the rabbit, unveiling previously inaccessible early aspects of NC in mammals.